Summary of work: Caloric restriction (CR) remains the only intervention that reproducibly extends lifespan, reduces the incidence of age-related disease, and retards aging processes in mammals. Although the effects of CR on aging have been widely studied in rodent models and other short-lived species, the relevance of this intervention to human aging has not been established. In 1987, the NIA began the first controlled trial of CR in a primate species. Results from these studies have thus far demonstrated that it is possible to initiate long-term CR (30 percent reduction from control levels) in primates and that physiological responses to the diet are similar to those reported in rodents. In addition, CR reduced risk factors for age-related disease, such as cardiovascular disease and diabetes. Summarized below are the most recent findings related to these ongoing studies. In the past year several studies have been completed relating to the physiology of aging in primates and rodents and effects of CR and other interventions. Additional use of MRI imaging in rhesus monkeys has repeated our earlier findings of an age-related loss in the volume of the striatum and putamen, two brain regions involved in motor performance, but no decline in overall brain volume. The data also suggested that this volumetric decline was attenuated in older monkeys on CR for over 10 years, but these results will need to be further evaluated at still older ages to confirm statistical significance. Analysis of learning performance in rhesus monkeys has also demonstrated age-decline in simple learning tasks, especially in a task that requires inhibition of an earlier response, and this performance decline also appears to be attenuated in female monkeys of CR. Investigation of several glycation products was conducted from skin samples obtained from the monkeys, and the results supported an age-related increase in this process with limited evidence that CR had retarded glycation thus far. Longitudinal evaluation of food intake has documented an age-related decline in food intake and motivation for food in male and female rhesus monkeys consistent to what is observed in aging humans. We found a negative correlation between food intake and the level of serum globulin. Further analysis of neuropeptide correlations with food intake is being conducted. We have also continued to monitor morbidity and mortality in our longitudinal studies of CR in rhesus and squirrel monkeys. The numbers of deaths and diagnosed diseases remain too low for conclusive statistical analyses; however, the emerging data continue to show lower morbidity and mortality in the CR monkeys in both species. In collaboration with the Laboratory of Cardiovascular Sciences, studies are underway to investigate the effects of CR on cardiovascular measures related to arterial stiffness in monkeys on diets with low and high salt content. Thus far, we have found that higher salt content increases arterial stiffness, but that this response depends on the ability of the monkeys to excrete salt in their urine. Two distinct populations of monkeys appear to be emerging?those that can handle large salt loads and those that cannot, which results in increased arterial stiffness. We are also nearing completion of a short-term (6?9 mo) study to determine if CR and 2-D-deoxyglucose (2DG), acting as a CR mimetic, can protect monkeys against the neurotoxin, MPTP, used to model Parkinson?s disease. We are conducting extensive physiological, behavioral, neuroimaging, neuromorphological, and neurochemical analyses to support the study. Results of experiments in various rodent models continue to demonstrate the effectiveness of 2DG feeding as a neuroprotective strategy; however, a recent mortality study completed in rats does not support the hypothesis that 2DG feeding will extend lifespan similar to the effects of CR. Indeed, long-term 2DG feeding appears to be toxic at certain doses that have provided neuroprotection. In a recent study in mice, we compared the neuroprotective effects of a CR regimen imposed daily versus CR imposed by every-other-day (EOD) feeding. EOD feeding was equally effective in attenuating brain damage induced by the glutamate receptor-mediated neurotoxin, kainic acid (KA), even though the EOD mice were eating near ad libitum levels at the time of KA treatment. These results suggest that it might be the adaptation to stress of a CR regimen rather than the degree of CR that is responsible for its neuroprotective effects. Other studies examining the insulin signaling pathway have indicated that the increased insulin sensitivity associated with CR is mediated through enhanced phosphorylation of the insulin receptor rather than through altered gene expression or protein production. Studies have also continued using an in vitro model of CR in which serum from CR rats and monkeys is applied to various cell cultures. Using a variety of stress inducers (heat, hydrogen peroxide, serum deprivation), we find that cells provided CR serum exhibit enhanced resistance and survival consistent with upregulated stress response genes and protein production that are observed. While insulin levels in the serum appear to be a major factor mediating these in vitro responses, use of microarray and proteomic technology is being applied to identify other important factors mediating the enhanced stress response associated with CR.